Assessment of μ-Opioid Receptor Signaling Responses for Fentanyl Analogs Using Luciferase Complementation Assay

To study the toxicity of fentanyl analogs that damage the liver and kidneys in rats, these analogs were evaluated by examining two types of μ-opioid receptor (MOR) signaling responses using HEK293 cells. The results indicated that, in the MOR-mini-Gi recruitment assay, the 50% effective concentration (EC₅₀) ranked as iBF < DAMGO ≈ 4F-iBF < 4Cl-iBF, and the maximum response (E_max) ranked as iBF ≈ DAMGO > 4F-iBF > 4Cl-iBF. In the MOR-β-arrestin 2 recruitment assay, the EC₅₀ ranking was DAMGO < iBF < 4F-iBF < 4Cl-iBF, and the E_max ranking was DAMGO > iBF > 4F-iBF. In addition, each of the desphenethylated metabolites, likely the major metabolites of these analogs, showed no MOR signaling responses.